DE2048375A1 - Making antibiotics effective - Google Patents

Description

Patent attorneys

Dr. Ina.Walter Abitz "' ^X ' ^October

Dr. Dieiar F. Morf
Dr. H * .ns-A. Browns "

8 Munich 86, Plenameuerstr. 28

MERCK & CO., INC., Hahway, New 'Jersey, V. St.A.

making antibiotics effective

The activity of antibiotics becomes effective through compounds made, which is the biosynthesis of the transport mechanism stimulate, which mediates the entry of the antibiotic into the cell. Method of finding such compounds and their use to potentiate antibiotic activity are intended. So are 'phosphonomycin, its analogs and derivatives by means of compounds which have the α-glycerol phosphate or the hexose-6-phosphate transport system by bacteria directly or 'after the metabolism.

Elimination of bacterial infections through antibiotic therapy is often caused by practical difficulties in achieving sufficiently high values of the. Thwarted antibiotic at the site of infection. In the fillings, as concentrations that are only effective on the hands are often used against antibiotics

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persistent organisms from the original, infecting stock. This problem is in the pall of the new antibiotic Phosphonomycin - ^ -) - (cis-1 ^ -EpoxypropyO-phosphonic acid / essential as it is rapidly excreted and its effects by common components of the plasma and urine, e.g. B. glucose or phosphate is counteracted. Furthermore, was found that the antibiotic occasionally opposed previously existing mutants that opposed this antibiotic are relatively persistent and occur in many bacterial populations, is ineffective. As a result, after Procedure to eliminate these difficulties in the antibiotic therapy wanted.

It is an object of the invention to provide a method for increasing the sensitivity of microorganisms to it Antibiotics to enable therapy with tissue values, which are available at appropriate doses. Another task is to prevent the occurrence of resistant ones Prevent bacterial organisms during antibiotic therapy. The invention also provides a method for Granting of sensitivity to bacterial populations, which otherwise either through earlier attainment of resistance to the antibiotic or due to internal insensitivity to the antibiotic cannot be treated. Another object is a method using appropriate triggers to create new paths and / or to increase the effectiveness of existing ways of taking up antibiotics by using suitable ones Triggers or inducers. Furthermore, according to the invention Method compounds are determined that act as inducers, which previously unexpected ways to • Tap into the absorption of antibiotics by bacteria. Furthermore There is an object in a method for increasing the α-glycerol phosphate transport path that is necessary for the uptake of

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Antibiotics is responsible and to tap into the Glucose-ö-phosphate transport route. Further tasks result from the following description of the invention.

According to the invention it was found that the activity of Antibiotics · can be made effective to a high degree by compounds that act to the effect that an existing one Is promoted away or new transport routes for bacteria are opened up. Compounds showing the activity of the α-glycerol phosphate transport system or De novo can cause a hexose-phosphate system in bacteria, so either before treatment with an antibiotic or used concurrently with an antibiotic to make the activity more active. For example are microorganisms that show no sign of sensitivity to either a specific antibiotic Reason for the absence of a transport route or because single there is a small amount of suitable transport routes compared to exposure to the antibiotic sensitized to inducers in order to bring about a suitable pathway or an increase in the to bring forth existing veges. The term "inducer" used here denotes a compound used, which either directly or after metabolism by host or bacterium with the regulating mechanism of the Cell interacts that regulates the biosynthesis of specific transport routes. In such cases where less than the maximum Synthesis speeds of the receptive transport system before introduction, the inducing agent into the Medium occur, the latter is considered to increase the rate of synthesis and thus the activity per cell of the transport system called increasing. In such cases, v / o the rate of synthesis of a particular class in the absence of

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inducers is practically zero, this has been the case Compounds referred to as opening up the latent potential of the cell while creating the transport pathway. The inducer does not have to, but is often a substrate for one of the group of proteins that it increases or opens up.

According to a special embodiment of the invention, ■ found that certain compounds act as inducers with bacteria either before or at the same time, with phosphonomycin, its analogs and derivatives acts and thereby makes the activity of these antibiotic substances effective. These .Compounds that act as inducers increase either the effectiveness of the α-glycerol phosphate transport route or open up a new transport route, e.g. B. a hexose phosphate transport system. Based on the effectiveness of these inducers for these transport systems, it was found that Organisms excited in this way have higher levels of phosphonomycin accumulate and thus be killed by relatively small doses of the antibiotic. Organisms that are no Signs of sensitivity to phosphonomycin either due to small amounts of α-glycorin phosphate system present or its absence in the organism is a result show a mutation to phosphonomycin resistance to this antibiotic by opening up the second hexose phosphate transport route made sensitive. Another advantage of the invention is that such Organisms that have two or more independent transport systems have a lower incidence of antibiotic Show resistance, since the simultaneous loss through mutation of two pathways in microorganisms is rare. There are too Process for finding compounds intended as inducers for these and other transport systems, giving access to phosphonomycin antibiotics to cells

_ 4. -

1 I.

BATH ORIGINAL 10 98 1 7 / 20.4 8

20Α8375

bring about, be able to work. Furthermore, the triggering of a third phosphonomycin transport route through eiboflavin> -5-phosphate demonstrated. It should be emphasized that those described here Inducers are not antibiotics or antimetabolites, but the biosynthesis of natural nutrient transport mechanisms stimulate, which mediate the entry into the cell of the phosphonomycin antibiotic. This Phenomenon applies only to the phosphonomycin antibiotics, since it was found that bridge strains induced in this way did not increase the sensitivity against other antibiotics tested. An important, beneficial consequence of using the 'Compounds serving as inducers consists in -that the inducer does not have to be present when the transport system allows the antibiotic to enter the bacterium convey. Thus, inducers can stimulate the transport of the phosphonomycin antibiotic if they are simultaneous present with this antibiotic, in competition with the transport proteins, can counteract well before the Administration of the antibiotic, given the opportunity to disperse in the host. ., ■ Furthermore, the invention requires. Procedure in contrast to the usual synergy by administering two Antibiotics · not that the blood level and the appearance! - speed of two or more components are matched to one another. Hence the one encountered so far Difficulty of actually resulting effective values of two different synergistic drugs On the basis of the present invention aur. switched, since the inducers used only the bacterium have to induce and then disappear; the transport proteins produced remain and thereby provide a means of entry for the antibiotic into the bacterial cell.

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According to another embodiment of the invention has now been found that certain polyhydric alcohols and their derivatives act as inducers either with an increase in the activity of the a-glycerol phosphate transport system or under Triggering a hexose-6-phosphate transport system act and therefore with phosphonomycin antibiotics in terms of Combat combating such bacteria. Thus, it was found that glycerin, a phosphatide and sugar phosphate are suitable as inducers. Sugar phosphates to be mentioned, which represent preferred embodiments of the invention, are glucose-6-phosphate, fructose-6-phosphate, Mannose-6-phosphate, glucose-1-phosphate, 2-deoxy-elucose-6-phosphate, 2-Aniino-2-deso: jcy-glucose-6-phosphate, glucose-1,6-diphosphate, Galactose-6-phosphate, bibose - ^ - phosphate and Like. Venn so sensitive bacteria with these. Sugar phosphates are brought into contact with a phosphonomycin antibiotic either before or at the same time, do so Phosphates or a metabolite derived therefrom effectively reduce the activity of the antibiotic, and it is then possible to lower it Use amounts of the antibiotic than would otherwise be necessary to combat the pathogen. This observed interaction in inducing a hexose-6-phosphate transport system in fact making the effectiveness of the phosphonomyein antibiotic effective is remarkable and completely unexpected. For example, it was found in tests with mice against E. coli that when using a Combination of glucose-6-phosphate and the antibiotic die dose of phosphonomycin antibiotic necessary to protect half of the mice less than one tenth of the amount of antibiotic alone required.

Instead of using a sugar phosphate, it is also possible to use inducing agents in situ by injection

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20A8375

14127 ^

Generate enzymes or substrates in the host. For example the hexokinase can be of microbial origin, from yeast, animal or those of human origin in tissue fluids any free ATP and glucose found to produce glucose-6-phosphate. Utilize «Preference is given to human hexokinase, Glycerokinase or glucokinase is used to lessen potential problems of antigenicity. It is also possible, simultaneous injection of a suitable, high-energy phosphate donor such as adenosine 5'-triphosphate or phosphoenolpyruvate with these enzymes and the ingestion or injection of suitable sugars accept s, such as for example fructose, mannose, glucosamine, which in their phosphorylated form induce the transport systems are to be used. Groups of enzymes can also be administered which ultimately have the inducers listed. medium, e.g. glycogen phosphorylase, which when exposed either on host-derived or indicated glycogen initially generate glucose-1-phosphate, which is produced by Effect of the endogenous or added phosphoglucomutase is converted into glucose-6-phosphate. The usefulness any combination of enzymes and substrates can thereby be identified (as a practical alternative compared to the direct test in infected animals or humans), in that these substances are animal or human J. Plasma can be added, incubated at body temperature for various periods of time and then the incubation mixture (after inactivation of the enzyme) 'in terms of its ability to target phosphonomycin-resistant microorganisms to make sensitive to phosphonomycin to test. Such a highly sensitive test system v.drd in the following described. An alternative system for studying enzymes or substrates or combinations of both, that with regard to the production of inducers of the phosphonomycin transport in si tu / best ent in the injection

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127 I.

tion of these substances in healthy animals or humans and then taking blood samples to test the effectiveness in the test system described below. In assaying these samples it is essential to first remove the blood cells and heat inactivate the plasma to eliminate any enzymes of natural or injected origin that produce inducers on the test plate itself rather than in the injected host. Methods and systems for the determination and classification of inducers of the two considered pathways of phosphonomycin entry into bacteria (the α-glycerol phosphate transport system and a hexose-6-phosphate transport system) are listed in detail. These methods and systems can easily be generalized for use in determining other useful inducers of these and heretofore undetected routes of transport with any strain of bacteria to be treated with phosphonomycin, its analogs and derivatives. This general method consists in the Isolation of a phosphonomycin-resistant mutant weed strain from a sensitive example of the strain to be treated and in the plague that the resistance is actually due to a failure of the phosphonomycin to penetrate the cell in adequate quantities. This loss of wild transport can be determined by either the simultaneous loss of the α-glycerol phosphate metabolic capacity of the mutant strain is detected or a reduced level of phosphonomycin is found even in these mutants when they are exposed to the drug-containing medium. These mutants are then either in brought a liquid or solid medium, the much higher phosphono-. mycinmengen fenthält as a medium in which the * ^ From angsetamm would survive, but has such a quantity that the mutant strain still dee growth allowed. Under

BATH

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Any arbitrary measure of induction will be added to these conditions Pathways of penetration for phosphonomycin as inhibition The bacterial growth turns into sensitive data because of the high levels of antibiotics surrounding the mutant. Induction can either be through inclusion of the potential inducer along with phosphonomycin in delicate slices or by distributing the inducer through the agar or liquid medium will. In those cases where it is expected that the inducer itself will be used with the drug If the new entry method competes (e.g. in the case of an α-glycerol phosphate itself), one can also get the vaccine from mutant organisms the inducing agent before their Exposure to combination with phosphonomycin, then exposing the competing inducer by some cell separation technique (Filtration, centrifuge and agl.) Is washed out, and these pre-induced cells with phosphonomycin in liquid medium can be combined. By identifying new pre-bindings that inhibit growth can result, it can easily be demonstrated whether the compound used itself is the growth inhibitor results in media which have no added ^ phosphonomycin contain.

The interaction of the inducing agents described here, with the phosphonomycin antibiotic provides a valuable means of combating and eliminating bacteria that ' otherwise are red atent to the action of a phosphonomycin antibiotic. So it can be a combination of the Inducer and antibiotic in a suitable carrier, which can be prepared by known methods, used topically to treat infections. The inducer and antibiotic can also be administered parenterally or orally to an infected animal or human

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single host either separately or in combination in a suitable pharmaceutical carrier, or one can be administered parenterally and the second can be administered orally. These pharmaceutical forms of the antibiotic and / or the inducing compound can be prepared according to known methods using suitable pharmaceutical solid or liquid diluents can be produced. The masses can be in the form of tablets, powders, granules, Capsules, suspensions, solutions, elixirs, syrups or other dosage forms that are particularly suitable for the oral administration are suitable. The mass can also be in the form of sterilized solutions or suspensions for parenteral use en administration. In these products, the sterile vehicle can be a sterile solution or suspension. The preparations containing the antibiotic can be mixed with solid diluents and / or tabletting aids, z. B. corn starch, talc, stearic acid, magnesium stearate, Rubber types and the like., Are mixed. The usual EL encapsulation or tableting materials suitable for the manufacture of pharmaceutical products can be used as long as they are not incompatible with the antibiotic or inducing compounds. These dosage forms may contain 25 to 500 mg of the active substance and can be administered in dosages that Can be given 1 to 6 times per day, depending on the age and condition of the patient, the infection and the route of administration. . ·

The term "phosphonomyein antibiotic" as used herein includes phosphonomyein and its derivatives of the formula

CH - OH P ^

(I)

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and the corresponding analogs of the formula

CH ₂ -C P <(II)

a, wherein A is hydrogen or a lower alkyl radical, X. Oxygen or sulfur, X and Z, which can be the same or different, the best

OH, OR, -HR-Bp, -HR-CH-COOH, -MOB, -HRHB ^ B ,,, NR XX.

-NB-C-NR _n B ₂ , -BHC-XB, HH-C-HB ^ B ₂ , -NCX, or -N ₃ , where X is oxygen or sulfur, B is a hydrogen atom, a hydrocarbon group Q is the substituted hydrocarbon group and B ₁ * and B _{2 represent} hydrogen, an acyl or a hydrocarbon radical or a substituted hydrocarbon radical. The formulas I and II also include the inorganic and organic salts of those compounds in which Y and / or Z represent a QH group, and the cyclic derivatives in which Y and Z are linked via a polyfunctional hydrocarbon compound , e.g. B. a straight-chain or branched-chain alkyl ene, aralkylene and aryl enpolyamine and amino alcohol radical and the like., For. B. Äthylendinmin, monoethanolamine, phenylenediamine, haphthalenediamine, o-aminophenol and the like., And those cyclic derivatives in which the grouping R..Ra is the remainder of a cyclic primary or secondary * amine, z. B. horpholine, piperidine or pyrrolidine.

If R, B ^. or B ₂ in formulas I and II is a hydrocarbon radical or a substituted hydrogen carbonate

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11-127 ■. / J

represent rest, this radical can be an aliphatic, cycloaliphatic, be an araliphatic, aromatic or heterocyclic radical which is optionally further substituted can be. Thus, for example, it can be an aliphatic Be rest, e.g. B. a substituted or unsubstituted alkyl. Alkenyl or alkynyl radical. Examples for R, JL and Rp, which represent an Araliphatic iiest, are such Cases in which the radical is an aralkyl or substituted aralkyl radical, for example benzyl, phenethyl, · Phenylpropyl, p-halobenzyl and o-, m- or p-alkoxybenzyl, Nitrobenzyl-, Amiiiophenäthyl-, Pyridylathy 1-, Nitrofurylmethyl, thienylpropyl radical and the like.

R, R ^ and R _{2 also} represent an aryl radical or substituted aryl radical, e.g. B. phenyl, naphthyl or substituted phenyl radical. ·,

According to the above, the amide group can or the amide groups can be derived from compounds that are themselves antibacterial. Examples of such compounds include 6-aminopenicillanic acid, 7-aminocephalosporanic acid, sulfa compounds, such as SuIf anil amid, Siilfadiazin, Sulphamerizin, Sulphamethazine, Sulfadimetin, ßulfapyridine, sulfathiazole, sulfisoxazole, Thiodiazole, sulfacetamide, sulfaguanidine, sulfachinoxaline, and p-aminophenylsulfonamide and p-aminobenzone sulfonic acid, antibiotic agents, such as ampicillin, streptomycin, Dihydrostreptomycin, cycloserine, cephaloglycine, cephalixin, and the like can be mentioned.

Those compounds of the formulas I and II which are acidic, i. H. the free acids can form salts, and these Salts represent a preferred embodiment of the invention

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because they are more stable than the free acid. As is apparent to the person skilled in the art, the compounds of the formulas I and II, where at least one of the radicals Y and Z is an OH group, form organic and inorganic salts, and both become. contemplated by the invention. Examples of these 'salts are inorganic metal salts, e.g. B. the sodium, aluminum, potassium, ammonium, calcium, magnesium , silver and iron salts. Organic salts which can be mentioned as examples include the salts with primary, secondary or tertiary amines, for example monoalkyl amines, dialkyl amines, trialkyl amines and nitrogen-containing heterocyclic amines. Typical examples are salts and amines, e.g. B. oc-phenethylamine, diethylamine, quinine, brucine, lysine, protamine, arginine, procaine, ethanolamine, morphine, benzylamine, ethylenediamine, Ν, Ν'-dibenzylethylenediamine, diethanolamine, piperazine, dimethylaminoethanol, 2-amino-2-methyl-1 -propanol, theophylline, esters of amino acids and N-methylglucamine B. erythromycin, gleandomycin or novobiocin ·, 'be.

The monoamide monoester derivatives and especially those Compounds which have a labile ester substituent are particularly valuable derivatives. With the here available When the term "labile ester" is used, a group is encompassed which can easily be biologically hydrolyzed, for example by Enzymes in the body fluids of living things including. lent people to the free acid or a salt thereof too that is more effective than an antibiotic agent. The amide group or substituted amide group present in the amide ester derivatives can also be easily incorporated into the Body fluids are biologically hydrolyzed, and therefore the amide-labile ester derivatives are used in antibiotic therapy suitable.

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14126 ■ Λ

The esters which are sufficiently labile for use as antibiotic agents are easily determined experimentally, e.g. B. by incubation with body fluids to determine whether or not the ester group is split off under these conditions. Other methods, including chemical testing, can also be used to determine whether particular ester groups are sufficiently labile. The esters which give detectable antibiotic activity after heating in an aqueous medium for about 2 hours at 37 ° C. and a pH of 2.2 or in an aqueous medium for 80 hours at pH 9 can be regarded as labile esters . Suitable labile ester groups to be mentioned are Xther of the formula -CHpOH ₁ a phenacyloxymethyl group, acyloxymethyl group of the formula -CIL) OA, in which A represents an acyl group which has an organic radical derived from an organic acid by removing the hydroxyl group, amide and substituted amide -Derivatives of these acyloxymethyl substituents, acylaminomethyl groups of the formula -CBUMHA, in which A has the meaning given above, thiomethylether. of formula -CHpSH, a Athinyloxygruppe of formula -CHpOCeCH, substituted Äthinyloxygruppen of the formula -CH ₂ OC = CR, a Vinyloxymethylgruppe of formula substituted Vinyloxymethylgruppen of the formulas - ₂ or -CH ₂ OCHbCRR or a nitrooxy group of the formula -CH ₀ ONO _0th In each of the above-mentioned formulas, R is a hydrocarbon radical or a substituted hydrocarbon radical as defined above.

Specific examples of such labile ester groups include the following: methoxymethyl-, tetrahydropyranyl oxomethyl, phenacyloxymethyl,, acetoxymethyl, butyryloxymethyl, Isobutyryloxymethyl, pivaloyloxymethyl,

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1412? · IS

Benzoyloxymethyl-, 2-methylbenzoyloxymethyl-, 2,6-dimethyl- 1-benzoyloxymethyl-, 2-methyl-6-chlorobenzoyloxymethyl-, 3-trifluoromethylbenzoyloxymethyl-, 2-nitrobenzoyloxymethyl-, 2-methylthiobenzoyloxymethyl-, 2-methyl-2-thiobenzoyloxymethyl-, 2-methyl-2-thiobenzoyloxymethyl- -Furylcnrbonyloxymethyl-, J-Pyridylcarbonyloxymethyl-, Pyrazinylcarbonyloxymethyl-, 2-Methylcyclopentylcarbonyloxymethyl-, I-adamantylcarbonyloxymethyl, Phenylsulfonylmethyl-, Phosphonoxymethyl- "Diäthylphosphonoxymethyl-, Carbäthoxyoaqnaethyl-, carbamoyloxymethyl, N-Methylcarbamoyloxymethyl-, Ν, Ν-Dimethylearbamoyloxymethyl-, Plienylsulfamoyloxymethyl, acetaminomethyl, benzoyl & minomethyl, methylthiomethyl, phenylthiomethyl, vinyloxymethyl, i

I-methylvinyloxymethyl and nitrooacymethyl groups.

The following examples illustrate embodiments of the invention.

example 1

Influence of the combination of glycerine or DL-cc-glycerine phosphate with phosphonomycin for its inhibition of various Bacterial strains

Overnight cultures of the indicated strains in nutrient solution (Difco) were diluted 10O-fold and an aliquot ύ of 0.05 ml was wiped over the surface of a 2 mm deep layer of the indicated solid growth medium in 50 cm "Petri dishes. Sensitive disks from a 7 mm diameter filter paper circle containing either 5 or 50 mg of phosphonomycin with an additional amount of glycerol or disodium DL-α-glycerol phosphate were placed on the surface of the inoculated agar.

After 18 hours of incubation at 37.degree. C., zones of inhibition became measured. The results are shown in the following table:

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1 ^ Ύ £ ( medium / i • P. A. Zone prices
mm diameter
BCD 10 0 tribe Nutrient agar / ε P 11 45 40 13th 0 E; coli
MB. 2489 Nutrient agar VJl 13th 13th 12th 10 0 E. coli
MB. 2498 Nutrient agar 30th 8th 47 43 16 0 E. coli
MB. 2489 A2 Nutrient agar . 5 12th 29 26th 15th 0 E. coli
MB. 2017 Nutrient agar 5 15th 40 18th 16 0 Pseudo,
aeruginoea
T 9 Nutrient agar 5 18th 31 29 23 23 Pseudo,
aeroginofia
T 19 Mueller Hinton
Agar 30th 21st 25th 23 16 Pr. Mirabilis
T 10 Nutrient agar '+ 10 5
Horse serum 5 10 15th - 15th D. pneumoniae
I 37 Brain heart
Infusion + 10 S
Horse serum 30th 12th 14th 14th D. pneumoniae
I 37 Brain heart
Infusion + 10?
Horse serum ό
30th 10 13th 18th 0 D. pneumoniae
I 2483 Brain heart
Infusion + 10 y
Horse serum ' 30th 15th 19th 13th 0 Strep. Pyogenic
nes
3009 ' Brain heart
Infusion + 10 5
Horse ser-um έ
30th 14th 17th 12th mm Strep, pyoge-
nes
1685 Brain heart
infusion i
30th 11 19th 15th mm SaI. bulkhead
muelleri 1814 Brain heart
infusion 30th 15th 19th 19th SaI. typhimur-
ium MB 1995 Brain heart
infusion 30th 19th 22nd Sal typhosis.
2866 30th

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14127 lh

Key as to the identity and amount of the to the Disc in combination with phosphonomycin added potentiators: '' ·

P - ■ Phosphonomycin (disodium salt) (quantities in the left column specified).

A - glycerin, 10 mg

B - glycerin, 1 mg

C - DL-α-glycerol phosphate, disodium salt, 10 ag D - DL-α-glycerol phosphate, disodium salt, 100 µg

It can be seen that glycerol acts synergistically over a broad spectrum λ of strains and only in the pail of E. coli 2498 (a mutant derivative of MB 2489), which is known to have no α-glycerol phosphate transport activity, and Proteus mirabilis (T 10) fails'. The latter strain, together with all of the sensitive Proteus species tested, has a very effective a-glycerol phosphate transport system, which is most likely constitutive, ie not subject to further induction.

Some significant examples of increased sensitivity are observed at the low level of added disodium DL-cglycerol phosphate, even though it is a known one. "At least% MB 2489 is the inducer of the transport system. Some antagonism is detectable at the high value (100 µg) added to the disk. This phenomenon is believed to represent the expected competition between phosphonomycin and α-glycerol phosphate for their usual transport system Glycerine, although an inducer, is not a substrate and therefore does not previously occupy the transport system whose activity it has stimulated.

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Example 2

Effect of glucose-6-phosphate on sensitivity of Escherichia coli and Staphylococcus aureus Phosphonomycin in liquid hedium of various compositions. .

Overnight liquid cultures were scaled to 1: 10,000 (1CK cells / ml) diluted in the specified hedium and combined with an equal volume of medium containing various amounts of disodium phosphonomycin. Me minimum inhibition concentration (H.I.C.) was the Final concentration of phosphonomycin, below that after one 24 hour incubation at 55 ° C turbidity was observed.

medium M.I.C. jag / ml Phosphonomycin Staphylococ Escherichia coli cus aureus HB 2017 MB 29 ^ 9 Mueller Hinton liquid 50 3.12 * ability (Difco) Mueller Hinton liquid 3 / I2 0.78 plus disodium gluc ο s e-6-phosphate, 25 / ig / ml Driving solution (Difco) 25th 12.5 Nutrient solution (Difco) 1.5 0.39 plus disodium glucose 6-phosphate

Nutrient solution (Difco) plus 5 % V / V defibrinated sheep blood ()

3.12

0.39

In both media it was found that glucose-ö-phosphate makes the susceptibility of gram-positive and gram-negative pathogens effective by a factor of 4 to 40. In nutrient fluid, the glucose-6-phosphate observed

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respected effect mimicked that observed with sheep leaf. Example 3

Influence of glucose-6-phosphate on the fraction of Bacterial stocks that survive at a given Phoephonomy'cin value ..

There are various dilutions of overnight KuItur-r solutions of the bacterial strains designated over the surface of Petri dishes wiped, which contained Mueller Hinton medium, 1.5% agar (Difco) and with the angege- λ surrounded values at Dinatriumphosphonomycin with or without 25 l / ml disodium glucose-6-phosphate were supplemented. From the number of colonies present at a specific dilution of the given organism, the number of surviving cells supplied is calculated for a given phosphonomycin value with and without glucose-6-phosphate, the values being given in the following table:

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%O

tribe iig / ml phos- '
'phonomycin Number of surviving Kolo
nienbildner per ml
Mueller Hinton Mueller Hinton
Agar alone agar plus
25 / its / ml GIu-
cose-6-phosphate 10?
10? 3 χ 1o2
5 χ 10 Escherichia
coli MB 2017 0
10 3 x
3 ^x 10 ^ ' 50 30th
100 · 3 x
3 x 10?
10? 3 χ 10 ^
. 3 χ 10?
<- 100
<10 Staph
coccus
aureus
MB 294-9 0
10
50
100. 3 χ
3 x
3 x
3 x 10 ^
10?
10? · · • Ji. f VJ £
1 χ 10?
5 χ 10p
5 χ io ^d Aerobacter
aerogenes
MB 3287 0
10
30th
100 7 χ
3 x
5 χ
3 χ 10 ^
; io 2 χ 10 ^
1 χ 10 $
1 χ 10?
<; io ' Staph
coccus
.aureus
MB 3036 0
10
30th
100 2 χ
2 χ
2 χ
< 10Z
io £ P ν 10
Ci Jv 1 V- * - »
1 χ 10?
1 x 10?
6 χ 10 Shigella
sp.
MB 3298 0
10
30th
100 2 χ
3 x
3 χ
1 χ

In all cases, a smaller proportion of the introduced bacterial population survives with the formation of colonies on the plate which contains glucose-6-phosphate than on the plate which does not contain this virus. In most cases the substantial remaining stock (on the order of 1 in 10 ^ to 1 in 10) that survives high levels of phosphonomycin is eradicated or largely reduced if glucose-6-phosphate is also present. A sensitization of the stock and the removal of resistant stock are obvious,

te

when this inducer is present.

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Example 4

Influence of glucose-6-phosphate on the size of the inhibition sz one that surrounds the delicate slices that this sugar phosphate in combination with phosphonomyein contain

Overnight cultures of the indicated strains grown in Difco broth were diluted 10X and a 0.05 ml aliquot wiped over the area of a Petri dish containing 10 ml of Mueller Hinton agar (Difco). Sensitivity disks consisting of filter paper disks with a diameter of 7 nm and containing either 5 or 30 µl disodium phosphonomycin with or without additional 5 PS disodium glucose-6-phosphate were placed on the surface of the inoculated agar. Zones of inhibition were measured after incubation for 18 hours at 37 ° C.

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14127 it

Bacterial strain diameter of the zone of inhibition in mm

5 ug - 50 / « 5 hp - 30 yig

Pnosphonomycin * Pnosphonomycin plus

without glucose-6-glucose-6-phosphate phosphate -

Escherichia 11 16 20 24

coli MB 2017

Staphylococcus O 11 13 20 aureus MB 2949

Aerobacter _# 0 0 16 26

aerogenes

MB 3287

Staphylococcus. 0 30 .27 40 aureus MB 3036

Shigella sp. O 10 24 37

MB 3298 "

The sensitization of cells by glucose-6-phosphate noted in the earlier example is shown here by means of a substantial increase in the zone of inhibition that surrounds the discs, which contain a mixture of phosphonomycin and Contain glucos-e-6-phosphate. ' It is also noteworthy that that in all cases where zone enlargement in If the presence of glucose-6-phosphate is observed, it was found that the inhibited area was relatively free of the myriad drug-resistant colonies that surround a disk of phosphonomycin itself. These Observations are in agreement with the development an alternating route for the entry of phosphonomycin ■ in cells that have lost their normally expressed α-glycerol phosphate transport route by glucose-6-phosphate. . *.

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Example 5

Example of a method for sieving phosphate stones as inducers for latent transport systems from Phosphonomycin in Escherichia coli

Escherichia coli MB 2498 strain is a subculture of mutant 6 described in Table I of the Journal of Molecular Biology, 21, 371 (1968). It lacks the ability to grow or accumulates on La-glycerol phosphate and is persistent against plhosphonomycin values up to 70 / Ug / ml in nutrient solution. (The wild parent strain is completely inhibited by 10 ug / ml disodium phosphonomycin.) * KB 2498 also has no alkaline phosphatase activity and decomposes exogenous phosphate esters to a minimal extent.

When looking for inducers for additional transportation. systems for phosphonomycin were '0.05 ml of a suspension

1 2

of 10 'cells / ml smeared over the surface of a 50' cm petri dish containing 10 ml drilling fluid, 1.5 % agar (Difco) and 25 µg / ml disodium phosphonomycin. Paper disks with a diameter of 7 cm and which could absorb 0.25 nal solvents were treated with solutions of various phosphate esters and applied to the agar surface. Zones of inhibition were genessen according I8etündi * eng incubation at 37 ° G.

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Tested connection

no
Disodium glucose-6-phosphate

Din atri um-fruc tο s e-6-pho sphat Disodium mannose-6-phosphate

Disodium 2-deoxy-glucose-6-phosphate

Disodium 2-amino-2-deoxyglucose-6-phosphate

Disodium ribose-5-phosphate monohydrate Phosphate / dylethanolamine

Tetrapotassium glucose ii ', ö'-dipho-sphatpentahydrate ,

Disodium glucose-1-phosphate

Dimagnesium-5-phosphoryl-ribose-ipyrophosphate-dihydrate

Disodium riboflavin-5-phosphate.

.2048375 hunt in the
'Disc Inhibition
nuigazone
ma through knife - 0 1.0
0.5
0.1 31
27
18th 10.0 42 6.0 31 1.0 27 25.0 "34 it 25 '35 30th 29 25th 34 25th 31 25th 25th 25th 14th

Among the compounds of the above experiment which showed no activity at a value of 25 jag per disc, were: inositol phosphate, adenosine 5 ¹ -phosphate, galactoee-1-phosphate, 2'-deoxyribose-1'-phosphate, aD-ribose-1-phosphate, ß-D-ribose-1-phosphate, aD-xylopyranose-1-phosphate, giucon-6-phosphate, hannose-1-phosphate, erythrose-4-phosphate, pyridoxine-phosphate, thiamine monophosphate, D-galactose-6-phosphate, D-fructose-1-phosphate, fructose-1,6-diphosphate, phosphoserine, phosphatidyl-choline, KjN-dimethyl-L-phosphatidylethanolamine and a large number of non-phosphorylifter tetroses, pentoses and Hexoses. The potentiation phenomenon thus shows a degree of specificity which, in the case of hexose phosphates, primarily includes such compounds.

- 24 -

BAD ΟΠ-C-NAL

109817/2048

14127

seem to close, which are generated by hexokinase, • and to which such hexose phosphates belong, which are known the glucose-ö-phosphate transport system (compounds 1, 2, 3 »4 · and 9).

None of the active compounds produced zones of inhibition at the levels tested when inoculated with MB 24-98 Plates consisting of nutrient solution / agar without MK 955.

Example 6

Influence of the combination of different phosphate esters on the inhibition of different bacterial strains

Overnight cultures of the specified strains in nutrient solution (Difco) were diluted 10X and an aliquot was made Amount of 0.05 ml over the surface of a 2 mm deep Wiped a layer of the specified solid growth medium. Sensitivity discs, consisting of circular filter paper discs of 7 mm in diameter which either 5 or 30 was disodium phosphonomycin with an additional amount containing the indicated phosphate esters were placed on the surface of the inoculated agar. Zones of inhibition were measured after incubation for 18 hours at 37 ° C.

- 25 -

109817 / 20Λ3

14127 P. U 25th 25th 20th 30th 22nd 21st 18th chme
Trains
H " ss e
set
I. tribe v, I 10 10 10 10 21st 10 11 15th 22nd MB 2489 VJl 33 33 26th 24 21st 31 26th 10 10 MB 2489
A2 30th 12th 10. 10 12th 14th 10 11 21st #) MB 2498 30th Zone size mm major
(compare keys for the
phatester)
^ PABCDEPG 23 22nd 20th 17th 16 21st 19th 11 12th MB 24980 5 V 13th 15th 0 0 0 12th 0 17th 19th MB 2017 5 8th 20th 20th 0- 0 01 .18 13th 0 O 0? 14th .30 13th 16 15th 15th 15th 12th 15th 16 0 14th T 27 5 11 23 23 23 ' 23 22nd 22nd 22nd 16 16 T9 5 12th 17th 17th 17th 15th 18th 15th 17th 23 23 T 10 30th 0 17th 15th T 19 . 0 15th 21st 18th

Bowl as to the type and amount of slices to go with Phosphate ester added together with phosphonomycin:

P - disodium salt of phosphonomycin (amount indicated in the left column)

A - glucose -6-phosphate, 5 J * g

B - 2'-deoxy-glucose-6-phosphate, 5 J * g

C - ribose-5-phosphate, 26 ng D - Thosphatidyl-ethanolamine, 25 WS E - Riboflavin 5-phosphate, 50 µg P - fructose-6-phosphate, 5> Ug G - mannose-6-phosphate, 5 pg H - 2-amino-2-deoxy-glucose-6-phosphate, 25 W5 I - glucose-1-phosphate, 25 ^ g

The MB 2489 strain is an Escherichia coli strain that grows well on α-glycerol phosphate and D-glucose-6-phosphate. It gives / ^ hriquid agar massive sensitivity to phosphonomycin, which runs through the whole legs of the hexose phosphateeter (A, B, P, G ₁ I), of which it is known that

, - 26 -

109817/2048

they induce the glucose-ö-phosphate transport route, is increased significantly.

The strain MB 2489 A2 was increased from the periphery of the Inhibition zone, which is a phosphonomycin (5 Με) and Surrounding disc containing glucose-6-phosphate (25 J * g), isolated. It was found to grow well on oc-glycerol phosphate, but not stimulated by growth Glucose-6-phosphate yields. Although this strain is just as sensitive to phosphonomycin alone as the parent strain MB 2489 is "(as expected, since its α-glycerol phosphate transport system is effective), it fails with regard to the stimulation of the nutrient solution agar by any of the Hexose phosphate inducer of the 'glucose δ-phosphate transport system. Furthermore, it results from the failure of the ribose-5-phosphate and phosphatidylethanolamine with regard to a synergistic effect, .that these esters also use the glucose-6-phosphate transport system induce. However, suggests that hiboflavin-5-phosphate is still effective on the presence of a third inducible pathway that mediates increased phosphonomycin transport.

Strain HB 2498 is a mutant of MB 2489 that does not have an α-glycerol phosphate pathway (ie it does not grow on ™ ^a -glycerol phosphate, but retains the inducible glucose-6-phosphate system). Although less sensitive to phosphonomycin to agar solution than MB 2489, it retains the ability to be stimulated by the phosphate ester inducers.

The strain MB 24980 was isolated as a resistant colony from a plate containing 25 µg / ml phosphonomycin and 25 µg / ml glucose-6-phosphate contained. In accordance

- 27 - -

.109817 / 20/4 8

with the above results, its sensitivity was increased Agar nutrient solution compared to phosphonomycin solely through riboflavin 5-phosphate increased.

The strain MB 2017 is an Escherichia pathogenic for mice coli. It gives extensive sensitization to agar- ' Nutrient solution through the entire class of phosphate ester inducers.

The strain T 14 is a Klebsiella species that was isolated from the urine of a patient immediately prior to initiation of phosphonomycin therapy; T 27 is a Klebsiella species isolated from the urine of a patient who had been on oral phosphonomycinr therapy for 7 days. Both strains are resistant to phosphonomycin itself on agar nutrient fluid, but show moderate sensitization in the presence of a large number of inducers of the glucose-6-phosphate vege.

The strains T 9 and T 19 are strains of Pseudoinonns aoruginosa, isolated from the urine of infected people. The trunks showed no noticeable sensitivity Agar nutrient solution versus the above phosphate esters.

The strain T 10 is a Proteus mirabilis strain, which aua dem Urine from an infected human had been isolated and showed no noticeable sensitivity to Mueller Hinton Agar versus any of the above phosphate esters.

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10981 7/2049

Example 7

Influence occurred in various growth media from. Glucose-6-phonphat on the size of the inhibition period, which the awareness disks that make this «uckerphoRphat in combination with phosphonomycin, exercise

An overnight culture of Escherichia coli MB 2489 which grew on nutrient solution was diluted 10Ofach, and an aliquot of 0.05 ml was deep agar medium swabbed over the surface of 2 mm, consisting of either nutrient broth, 1.5 '$> Agar (Difco), brain-heart infusion, 1 »5 i» agar (Difco), Mueller Hinton agar (Difco), trypticase-So, yes agar (BBIi) or a "human urine agar". I) a3 The latter medium was prepared by centrifuging urine collected from adult males immediately after sleep, filtering the supernatant material through a membrane to achieve sterility, and using Filtra with a tenth volume of autoclaved 15? 3n. Eoble agar (Difoo) was combined in water to create a solid medium. Sensitivity disks made from a 7 mm diameter filter paper disk containing 5 or 30 μg dintrium phosphonoraycin with and without disodium glucose-6phonphate were placed on the surface of the inoculated agar. Zones of inhibition were measured after 18otündiger Inkubierung.bei 37 ⁰ C.

- 29 -

': ■■ "· Cr.; GiNAL

10 9.8 1 7/204 p

14-127 30

Medium used Diameter of the zone of inhibition

(mm)

5 »g 30 / ig
Puosphonomycin
alone 24 • 12th 5 / g 30 wc
PhOsphonomycin plus
Glucose-6-phosphate
(25 ^ g) 33 Nutrient solution. 12th 15th 28 26th Mueller Hinton-
solution O » 19th 20th • 20th Brain heart
infusion O 16 24 Trypticase Soy
solution O 18 - 26th Human urine 9 14th

The activity of phosphonomycin alone is related to the nutrient solution in that. other medium used clearly counteracted. This antagonism can lead to an over-

η Vi "F *

weighing extent aie. high levels of sodium chloride in Mueller Hinton solution, glucose and phosphate in brain-heart infusion and trypticase soy and phosphate ions in human Urine. This disadvantageous phenomenon is mainly caused by the inclusion of glucose-6-phosphate eliminated in the sensitivity disc.

Example 8

Effect of glucose-6-phosphate on sensitivity of Escherichia coli strains to different. Phosphonomycin analogs

.Ub ernacht culture of Escherichia coli, strain MB 2489 (which includes both the oc-glycerol phosphate transport system and possesses the glucose-o-phosphate transport system) and MB 2498 (which only has the glucoaiß-6-phosphate pathway and therefore relative to phosphonomycin alone

109817/2048

resistant) were diluted 10 times and an aliquot of 0.05 ml was wiped over the surface of a 2 mm nutrient solution of 1.5 % agar (Difco). Sensitivity disks made from a paper disk of 7 ″ in diameter, containing disodium phosphonomyein or one of the indicated analogues in the indicated amounts together with another 5 jig of disodium glucose-6-phosphate, where indicated, were placed on the surface of the inoculated agar. Zones of inhibition were measured after incubation at 37 ° C for 18 hours.

, I.

Effective substance

lot 5 " MB 2489 + G- MB 24 ^ 8 ng 2.5. no G- 6-P no G- + G- Γ 1.0 6-P 28 6-P 6-P 0.3 14th 25th 0 30th 500 12th 20th 0 24 50 0 18th 0 24 5 0 38 0 • 15th 500 20th 32 9 42 • 50 0 13th 0 36 · 5 0 35 0 16 16 26th 8th 38 0 10 0 29 0 0 12th

Phosphonomycin

Monodi eye1ohexylamine salt the 1-methyl-1 ', 2-epoxyä thy lpho sphonic acid

Di eye lohexyl ammonium salt 1,2-Epoxyä thy lpho-sphonic acid

It has been found that glucose-6-phosphate makes the sensitivity of phosphonomycin-susceptible and resistant strains effective, so that they now respond to weak analogs of phosphonomy to the same extent as to phosphonomycin itself. ·

- 31 -

109817/2048

o g -: -: g:. \ 'al

Example 9

Exposure to phosphonomycin and glucose-6-phosph; t and their combinations in the treatment of infected mice '

Female C.D.l '- mice of medium molecular weight of 22.5 g were administered intraperitoneally with 16-hour culture fluids, which were appropriately diluted in brain-heart infusion, infected. Pure E. coli contained the

speaking value 2.5 x 10 'cells or dosages of 7 ^ for Shigella 2.3 10 cells or doses of 3 At the time of infection, the disodium sala des Phosphonomycins and sodium glucose-6-phosphate administered separately in 0.25 ml subcutaneously at a separate location, one on each side of the RückAache. The results of this Experiment are shown in the following table:

Test organism ^ED

50

G-6-P disodium- DSP + DSP + 0.1 mg

/ ig phosphonomycin 1.0 mg G-6-P

'(DSP) G-6-P

yUg % yUg %

Escherichia coli > 4000 155 100 12th 8th 91 58 2017 Shigella (118-57) > 4000 1000 100 82 8th 15OO 150 3303

- 32 -

BATHROOM OHIGiNAL

10981 7 / 20A8

14127 33

Example 10

Influence of phosphonomycin and glucose-6-phorpha.t as well their combinations in the treatment of infected homes

In further girls' tests, which were carried out as described in Example 1, with the exception that the antibiotic was combined with the sodium glucose-6-phosphate and in injection, the following results were obtained in mice infected with Aerobacter aerogenes and Staphylococcus aureus received:

f

Disodium phosphonomycin (DSP) sc test organism. 50

ττ?

ISP ". Ng G-6-P added to DSP alone WOO 1000 500 100 alone

det USAGE

aerogenic 3148 10 000+ 287 3 000 7 700 10 000> 4

Staphylococcus

eureus

Smith 2949 96 22 50> 4

Example 11 -

Influence of fructose-6-phosphate in the preparation of the virus of phosphonomycin in mice

The effect of fructose-6-phosphate in making it active the fight against experimental 'bacterial infections Phosphonomycin in mice was compared with that of glucose-6-phosphate in experiments according to the procedure of Example 10. The antibiotic was again with the sugar phosphate combined in a single subcutaneous injection for

-35 -

109817/2048

14127 '

Time of intraperitoneal inoculation with Escherichia coli HB 2017 administered. ·

Active agent dose cug) ED ₅₀ , ng phospho

nomycin

None "- 2000

Disodium glucose-6-. 1000 17

phosphate 600 31

300 69 *

100 470

Disodium fructose-6-1000 17

phosphate 600 - 57

3OO 202

100 534

It can be seen that fructose-6-phosphate is an extent exerts on efficacy against phosphonomycin, which is comparable to that which was previously used in the parallel experiments with glucose-6-phosphate was observed. This equivalency was increased from both the same in vitro observed inhibition expected in Example 5, if any of these sugar phosphates was combined with phosphonomycin, than for the safety of their conversion due to the sufficient phosphoglucose isomerase activity present in the plasma.

Example 12

Therapeutic effects of phosphonomycin taken orally infected mice that ingest glucose-6-phosphate either orally or subcutaneously

The procedure of Example 9 was used for the Pail from Escherichia coli repeated in 2017 with the exception that immediately after infection, the disodium salt of phosphonomycin was administered orally while disodium glucose-6-

'109817 / 2048-

phosphate, where indicated, either orally or subcutaneously Ways was administered. In no case was protection observed, when glucose-6-phosphate alone at the value of 4000 µg orally or subcutaneously in the absence of phosphonomycin was administered.

Glucose-6-way dose of orally administered phosphate phosphonomycin (ug), the 50%

lay the animals schüWt { ED)

O - 2000

1000. oral. 2000 100 subcutaneous 37.

Glucose-6-phosphate is an effective therapy potentiator for orally administered phosphonomycin (25-fold sensitization), when sugar phosphate is administered subcutaneously. No increase in sensitivity is observed when the sugar phosphate administered orally at this value.

Example 15

Therapeutic efficacy of phosphonomycin, which is administered parenterally to infected mice, -the oral glucose-6-phosphate salts ingestion, was administered

The procedure according to Example 9 was repeated for the case of Escherichia coli 2017 lait the exception that immediately after infection, the disodium salt of phosphonomycin was administered subcutaneously, while glucose-6-phosphate in of the given form orally by administration in 0.25 ^ l of water was administered. In no case was protection observed with administration of the glucose-δ-phosphate salts alone, Oral administration of 2.5 mg n-octylammonium chloride (without glucose-6-phosphate) alone caused a decrease

- 35 -

109817/2048

14127

would take the ED ^ Q value of phosphonomycin administered parenterally at the same time.- The n-octylammonium salts of glucose-6-phosphate were converted into the free acid by passing through a column containing a 20-fold excess of Dowex-50 (H ⁺ contained, subsequent neutralization of portions of the eluate with 0.7, 1.5 or 2.0 molar equivalents each of the free n-octylamine base and 1.5 »0» 5 or 0 molar equivalents of NaOH to achieve a pH of 7 , 5 produced in each case.

Glucose-6-phosphate salt (mg)

Dose of phosphonomycin, which protects 50% of the animals ()

Attempt I. no Experiment II no . . 500 * 827 100
50 5 ■ 27
65 125 Disodium
salt 25th 5 125 068 12.5 I '■' 502 714 6.25 551 10
1 66
502 Sodium 0.5
+ n-octyl-
ammonium 1.5 Di-n-octyl
ammonium Sodium 0.5 "~
n-octyl
ammonium 1.5 __ Sodium 1.5 ~~
n-octyl
ammonium 0.7 _

109817/20 4 B

■ "-TED

Orally administered glucose-6-phosphate makes the phosphonomyein therapy effective and becomes effective in this direction in proportion to the proportion of inorganic counterion that is passed through lipophilic amine is replaced, made even more effective.

Example 14

Making phosphonomycin therapy effective at the same time administered galactose-6-phosphate in case of staphy lococci infected mice

An investigation into the effect of galactose-6-phosphate regarding the effectiveness of the control of Staphylococtsen infections caused experimentally in mice by Phos- ■ Phonomyein 'was determined by applying the methodology described in Example 6 on this strain Finding justified that 25 J * g of this sugar phosphate, when added to a sensitivity disc containing 5 yUg of phosphonomycin, a depression zone of 21 mm, in contrast to the disc with no additional addition, with a zone of 17 mm, if the slices on one with Staphylococcus aureus Smith 294-9 ' inoculated nutrient agar plate. In the following Therapy attempt was the antibiotic with the sugar phosphaf combined in a single subcutaneous injection at the time of intraperitoneal injection with 10 cells each Mouse (14 IjDcq) administered with the cells in. Brain-heart infusion had grown.

- 37 -

109817/2048 '

14127

^ED 50

Potentiator Bo si s
(/ β) no ' ■ ■ * Disodium glucose e-6-
phosphate 4000 Disodium galactose
6-phosphate 4000

5 (ag. Phosphonomycin)

212 91 25

The effect of galactose-6-phosphate as a potentiator is explained by the proven existence of an inducible galactose-6-phosphate transport system in Staphylococcus. Galactose-6-phosphate is a metabolite of lactose hydrolysis unique to certain gram-positive organisms, but is not produced or used by E. coli. The failure of galactose-6-phosphate to make the phosphonomycin effect on E. coli can be traced back to this (Example 5)

Example 15

Effect of mannose-6-phosphate in terms of potency of phosphonomycin in mice

The effect of mannose-6-phosphate on the effectiveness of the control of experimental infections in mice by phosphonomycin was compared with that of glucose-6-phosphate in experiments according to the procedures of Example 10. Bas antibiotic was again titrated in combination with a series of fixed values of sugar phosphates in a single subcutaneous injection at the time of intraperitoneal Beimpf ung Escherichia coli MB administered 2017th ·

109817/2048

14127 dose
(/ 6) Potentiator _ no· 1000 Disodium glucose
6-phosphate 1000 Dinatri um-mannos e-
6-phosphate 500 Il 250 Il 125 Il

^ED

Gig phosphononiycin)

1420 18

19th

. 25th

92

490

Mannose-6-phosphate exerts a level of efficacy over phosphonomycin equivalent to its comparable Glucose-6-phosphate values, even though its effect in vitro is only a tenth of that of glucose-6-phosphate amounts to. This discrepancy can be due to the conversion of mannose-6-phosphate to glucose-6-phosphate in vivo sequential exposure to mannose phosphate isomerace and phosphoglucose isomerase, enzymes whose activities in plasma and in the walls are detectable by blood vessels.

Example 16

Making phosphonomycin therapy effective in mice by glucose-1-phosphate and eibose-5-phosphate

The effect of glucose-1-phosphate and ribose-5-phosphate regarding the effectiveness of phosphonomycin in controlling experimental bacterial infections in mice has been tested with that of glucose-6-phosphate compared according to the information in Example 10. The antibiotic, in turn, was considered to be beneficial in terms of its healing properties in combination with the specified fixed

- 39 -

1 0 9 8 1 7 /? Π / ♦ f.-

Zacker phosphate values titrated in a single injection at the time of intraperitoneal inoculation E. coli MB 2017 administered.

Potentiator dose _ ' _# EDcq (/ s) 1000. (ag phosphonomycin) no 1000 940 Disodium glucose
6-phosphate 1000 '5 ■ Dipotassium glucose
1-phosphate , 9 Disodium ribose
5-phosphate * 158

* In this sample, a specific test with glucose-6-phosphate dehydrogenase demonstrated that they by no more than a part per thousand glucose-6-phosphate was contaminated.

The potentiating ability of 1000 was bibose-5-phosphate, although significant, only that which is produced by about 100 lags of glucose-6-phosphate is equivalent (cf. Example 11). This degree of relative potency was "given by the weight ratio of ribose-5-phosphate to glucose-6-phosphate, which is equivalent to increased zones of inhibition generated in vitro (Example 5), prevented. the equivalent activities of glucose-1-phosphate and Glucose-6-phosphate in vivo, despite the differences in vitro, are most likely due to the rapid conversion of the 1-phosphate into the 6-phosphate through the action of phosphoglucomutase, which is known to be present in the plasma.

109817/2048

Example 17

Making Phosphonomycin Therapy Effective in Streptococci infected mice by co-administered lactose

After applying the methodology described in Example 6 to streptococci, it was found that 250 µg of lactose, which "were added to a sensitivity disk containing 30/6 phosphonomycin, a zone of inhibition of 27 mm in diameter compared with a zone of 12 mm in the case of a disc without an additive when the discs were placed on a nutrient agar plate inoculated with Streptococcus faecalis H. In the following therapy attempts 14 colonizing units (7 LDc-q) des pathogenic Streptococcus pyogenes (1934-) »the brain-heart solution grown and treated with 105 ml of horse serum, inoculated intraperitoneally. Simultaneously were 0.5 ml of either a lactose solution or a salt reference solution is injected subcutaneously, which is based on experiment I a single oral dose of 0.5 ml of phosphonomycin (by force administration) and four consecutive doses in Experiment II oral 0.5 ml doses of antibiotic after 0, 2, 4 and '6 Hours after infection.

Potentiator Dosxs'
(mg) (total administered
Phosphonomycin, iig) . 'Try I. no
Lactose 4th 3950
1530 no
Lactose Experiment II
4th 2100
800

- 41 -

109817/2048

Neutral saccharides can therefore reduce the phosphonomycin effect make or potentiate both in vivo and in vitro. Certain streptococci, together with ßaphylococci, also show inducible metabolism of lactose to galactose-6-phoBphat.

Example 18

Effect of phosphonomycin to protect mice, resulting in the absence of Induziermittels by bacterial mutant isolates which high Wert.e of La Glyceriiiphosphat transport system, were infected · -

Mutants were isolated from E. coli 2017 using a mutagenesis and mutant determination grid described in Biochimica et Biophysica Acta, Volume 60, pages 422 to 424, 1962, and these mutants showed high levels of oc-glycerol phosphate and glycerol metabolism without the need for " previous growth in the presence of these inducers, as shown, for example, with the natural strains. The diameter of the zones of inhibition around the sensitization discs, which contain 5 mg of phosphonomycin, which were applied to nutrient agar plates inoculated _{with mutants C 1, C 2 and the parent strain} , were 20, 24 and 12 mm, respectively. Since it was shown '(Example 1) that the addition of glycerol to these disks on the original strain increased the zone size from 26 to 29 mm, it can be concluded that the mutant strains contained levels of phosphonomycin Transport system (ie, the La glycerol phosphate transport system) similar to those of induced wild strains e are comparable. Therefore, the susceptibility of these mutants to phosphonomycin therapy should be predictable from the susceptibility of induced wild strains in other situations. Mutants Cy. and Cp of the parent strain were tested under identical conditions

- 42 -

109817/2048

cultured (described in Example 9) and inoculated intraperitoneally into mice at the given response values. Mice were injected subcutaneously with phosphonomycin immediately after infection.

• Bacterial strain number of vaccinated ED ₁ -Q

(Virulence) ty ^s phosphonomycin;

E. coli 2017 4.2 10 ⁶ 943

(Parent strain) (10 LD ™)

Mutasite C. 4.7 x 10 ⁶ 12

^Ί (30 LD ™)

(9

Mutant C ₀ 1.2 1 ⁷ 14

² C9 L)

Even though the mutants have full virulence, they are controlled by a remarkably low level of phosphonomycin *, which means that the uninduced wild strain in vivo develops far less than its full inducible capacity for responsiveness to phosphonomycin. It can be concluded from this that it is possible to establish maximum values of the La-glycerol phosphate transport system and a correspondingly more effective therapy with phosphonomycin in any clinical situation (e.g. intestinal tract or skin infections) where the absence of glucose is a problem effective induction by simultaneously administered potentiating agents (e.g. glycerine), which reach such places, ensures.

109817/20 ^ 8

Example 19

Effect of co-administered glucose-6-phoopha't on susceptibility to phosphonomycin, in vitro and in vivo a bacterial variant that shows resistance to phosphonomycin in humans during therapy had accepted

The strains of Escherichia coli used in the following represent, in the case of M 13, an isolate from the urine of an infected female animal immediately before sedative treatment with phosphonomycin, and in the case of M 21 -an isolate from the drug-resistant organisms present in the urine of this organism after 7 days of antibiotic therapy. The in vitro susceptibility tests were carried out in the manner described in Example 6. The mouse protection test carried out in vivo was carried out as described in Example 9 after intraperitoneal treatment with the specified number of organisms.

Sensitivity Tests · In Vitro

Zones of inhibition (mm) surrounding the discs containing 30 Jf ¹ S phosphonomycin alone or in combination ndt 5 J * S glucose-6-phosphate '

Phosphonomycin alone plus glucose-6-

phosphate

E. coli M 13 19 x 28

E. coli Μ 21 0 (less than 20

7 mm;

- 44- -

109817/2048

14127

Healing effects of phosphonomycin in infected mice . (ED ₅₀ in mg)

Phosphonomycin alone

E. coli M 13.
3.7 10 'cells

0.25

Phosphonomycin at the same time as 1 mg disodium glucose-6-phosphate administered

. 0.035

E. coli M 21
1.2 χ 10 'cells
- 10 LD ₅₀

17.5

2.5

* At the highest drug value administered to this group, 20 mg per mouse, only 3 eggs were infected five Animals protected. In the other three groups, complete protection was given to no more than the two times the mean stated value observed.

Since the resistant strain retains the ability to respond to phosphonomycin after the simultaneous addition of glucose-6-phosphate, it is concluded that the hexose-6-phosphate transport pathway is not appreciably induced by endogenous substances in natural urinary infections in humans. If transport is induced by the deliberate concomitant administration of inducers, resistance to phosphonomycin should be prevented or eliminated and therapeutic elimination of such strains would be enabled .

-45 -

'109817/204 8 ·

Example 20

Effect of simultaneous administration of Glueοse-6-phosphate on the sensitivity of a wild, inducible bacterial atom and one of the non-inducible ones derived therefrom Mutant © against phosphonomycin in vitro and in vivo

One with 201? A designated mutant, which gives no additional sensitivity in vitro to phosphonomycin after addition of glucose-6 ^ -phosphate, was derived from its parent strain, the naturally occurring pathogenic Eccherichia coli 2017, by the method described in Example 6 for isolating the strain MB 2489 A2 from his Starting strain MB 2489 described method isolated. The mutant 2017 A showed a normal ability to convert glucose-6-phosphate. Its in vitro sensitivity to phosphonomycin was determined twice in vivo in relation to the starting strain in the mouse protection experiments described below using the procedure listed in Example 9.

In vitro susceptibility tests

Zones of inhibition (mm) surrounding the discs containing JO »g phosphonomycin alone or in combination with 5/6 disodium glucose-6-phosphate ^/

Phosphonomycin alone plus glucose-6-

pho sphat,

E. coli 2017 18. 27

E. ooli 2017 A 17.5 18

109817/2048

14-127

Healing effects of phosphonomycin in infected houses

( ₀ in mg)

Attempt I.

E. coli 2017 1 χ 10 ⁶ cells

E. coli 2017 A 1 χ 10 ⁶ cells'

Experiment II E. coli 2017 5 χ 10 ⁶ cells

Pho sphonomycin alone

0.250

0.166

0.821

Phosphonomycin at the same time as 1 mg disodium glucose-6-phosphate administered

0.015

0.284 0.021

E. coli 2017 A 9 χ 10 ⁶ cells

- 55 LD ₅₀ .

1.420

1.150

The difference between the Mut ante 2017 A and the combination of glucose-6-phosphate with phosphonomycin in vitro is in vivo due to the failure of the co-administered glucose-6-phosphate to increase the effectiveness of Phosphonomycin in the treatment of this mutant infected muses. Thus, the normal activation of the medicinal properties of phosphonomycin is through Glucose-6-phosphate attributed to its direct action on inducible infecting bacterial strains and not by any responsiveness of the host that presumably

- 4-7 -

109817/20

could be tet (ζ. Β. increased drug absorption or immune response), which influences the mutant just as favorably would like the original stem. The same effectiveness of phosphonomycin alone against infections Reason for inducible and non-inducible organisms indicate that endogenous inducers are either absent or insufficient (in those areas of the body that are exposed to microorganisms is affected) to the biosynthesis of the hexose-6-phosphate transport system to evoke. The established Wirkixa, ^ - benefits resulting from the induction of this system, expressly require the administration of exogenous inducers by the therapist.

According to the invention, the bacterial transport systems

in infected animals using low doses of active agents, particularly by the oral route be brought about, if cleavage of the ester bond either by bacterial intestinal, liver, kidney, Plasma or urine phosphatases are avoided. The preferred Active agents are carbohydrate esters of the Formula:

Y 'CH ₀

ι tL

R ¹

wherein -CH ₀ - the terminal methylene group of the grouping R ¹ -CHp-OH, a pentose or hexose, such as, for example, glucose, fructose, mannose, 2-deoxy-glucose, 2-amino-2-deoxyglucose, galactose, ribose, a 1 -substituted ribitol, e.g. B. riboflavin, ader glycerin and Y 'the following groupings

- 48 -

1 0 9 8 17/2 0 U B

14127 W.

X "

Z X

-CH ₂ -]

-OSO ₃ H ₂ -SO ₃ H

-CH ₂ SO ₂ NHR ₁ or

mean in which. X, Y, Z, R ₁ and R _{2 have} the meaning given above and their salts

- 49 -

109817/2048

Claims (18)

W27 October 1, 1970 Patent claims 1. a method of making the activity of a phosphonomycin antibiotic effective, characterized by this, dacs Bacteria are brought into contact with an inducer, which increases an existing one Way or by opening up a new transport route for the bacteria acts and the bacteria spacer or at the same time brought into contact with a phosphonomycin antibiotic. 2. The method according to claim 1, characterized in that an inducer is used which is a hexose phosphate transport system can bring about. J. Ancestors according to claim 1, characterized in that an inducer is used which is a glucose-6-phosphate transport system can bring about. 4. The method according to claim 3 »characterized in that a sugar phosphate is used as the inducer. 5 · The method according to claim 3, characterized in that duss a phosphatide is used as an inducer. 6. The method according to claim 1, characterized in that an inducer is used which the α, -glycerol phosphate system can increase. 7. The method according to claim 6, characterized in that a polyhydric alcohol is used as an inducer. - 50 - 1 098 17/2048 8. The method according to claim 1, characterized in that that used as an inducer glucose-6-phosphate will. 9. The method according to claim 1, characterized in that that mannose-6-phosphate is used as an inducer. 10. The method according to claim 1, characterized in that glucose-1-phosphate is used as the inducer will. . 11. The method according to claim 1, characterized in that that ribose-5-phosphate is used as an inducer. 12. The method according to claim 1, characterized in that that use galactose-6-phosphate as an inducer will. 13. The method according to claim 1, characterized in that that lactose is used as an inducer. 14. The method according to claim 1, characterized in that that a phosphonomycin salt is used as a phosphonomycin antibiotic. 15 · Procedure for determining inducers for the Entry of a phosphonomycin antibiotic transport system in bacteria, characterized in that a phosphonomyein resistant mutant in a medium is grown which is a phosphonomycin antibiotic and a test compound, contains and such compounds - 51 - 109817/2048 14127 η fertilizers can be chosen as inducers, which the Inhibit growth of the mutant. 16. Method of Enabling the Activity of a Phosphonomyein Antibiotic in Antibiotic Seeing Therapy, characterized in that an inducer of a phosphonomycin Transpqrtweges in the infected Bacterium is given before or at the same time as the phosphonomycin antibiotic. 17. The method according to claim 10, characterized in that that the inducer coincides with the phosphonomycin antibiotic by parenteral administration is administered. 18. The method according to claim 10, characterized in that the inducer parenteral and the phosphonomy an antibiotic administered orally. An antibiotic composition characterized by a phosphonomyein antibiotic, an inducer of a phosphonomycin transport system in bacteria and a pharmaceutically acceptable carrier. OPTIMAL JNSPECTED - 52 - 109817/2048